Stem cells are rare cells located in specific niches where they are maintained in a quiescent state (Spradling et al, 2001; Lavker et al, 2000). Stem cells have been defined as cells that have the ability to perpetuate themselves through self-renewal and to generate mature cells of a particular tissue through differentiation. Although one would assume that each tissue arises from a tissue-specific stem cell, rigorous identification and isolation of these somatic stem cells has been accomplished only in a few instances.
Stem cells in organs other than the prostate have been identified by their expression of specific antigens, such as stem cell antigen-1 (Sca-1), alpha 6 integrin, and Bcl-2. The present inventors have determined that these antigens can be used to identify the stem cell population in the proximal region of ducts. Sca-1 is expressed on the surface of stem/progenitor cells from a variety of murine tissues, such as hematopoietic (Spangrude et al, 1988), cardiac (Matsuura et al, 2004), mammary gland (Welm et al, 2002), skin (Montanaro et al, 2003), muscle (Asakura, 2003) and testis (Falciatori et al, 2004). Alpha 6 integrin (CD49f) is expressed on the surface of primitive cells in the liver (Suzuki et al, 2000) and skin (Tani et al, 2000). Anti-alpha 6 integrin antibodies have been used to enrich for spermatogonial stem cells from mouse testis (Shinohara et al, 1999). Bcl-2, an intracellular anti-apoptotic protein (Adams et al, 1998), may protect primitive cells from death and is expressed by hematopoietic, keratinocyte and colon stem cells (Domen et al, 2000a; Potten et al, 1997; Tiberio et al, 2002). The expression of CD133 (prominin) has been found on human putative prostatic stem cells (Richardson et al, 2004). Signaling molecules such as Wnt and Notch are also involved in stem cell renewal and stem cell niches (Walsh et al, 2003). Notch1 expression has been noted in prostate epithelial cells during normal development and in prostate cancer cells (Shou et al, 2001).
The most definitive evidence for stem cells' definition is their ability to reconstitute an organ. Serially transplanted bone marrow can reconstitute lethally irradiated mice (Chen et al, 2000; Maggio-Price et al, 1988), and the number of successful serial transfers depends on the size of the grafts and the time intervals between transfers (Jones et al, 1989).
Cell surface molecules on various types of cells are given a cluster of differentiation (CD) designation in which each CD molecule designation describes a surface molecule (marker) identifiable by a cluster of monoclonal antibodies that display the same cellular reactivity. CD designations are assigned at regularly held international workshops on human leukocyte differentiation antigens. For example, the CD19 marker is specific to B cells, and the CD33 marker is specific to myeloid cells. At the present time, it is not known how many of the markers associated with differentiated cells are also present on stem cells.
Cancer is caused primarily by an increase in the number of abnormal cells derived from a given normal tissue, invasion of adjacent tissues by these abnormal cells, and lymphatic or blood-borne spread of malignant cells to regional lymph nodes and to distant sites (metastasis). Pre-malignant abnormal cell growth is exemplified by hyperplasia, metaplasia, or most particularly, dysplasia. The neoplastic lesion may evolve clonally and develop an increasing capacity for growth, metastasis, and heterogeneity, especially under conditions in which the neoplastic cells escape the host's immune surveillance.
As understanding of the pathophysiological role of cancer increases, the role of both tumor markers and genetic information becomes more important in the management and treatment of cancer patients. Tumor markers are substances that can be measured quantitatively by biochemical or immunochemical means in tissue or body fluids to detect a cancer, to establish the extent of tumor burden before treatment, to diagnose as aides in staging or confirmation of histopathology, to predict the outcome of drug therapy, and to monitor relapse. Measurement of tumor markers has been used to screen total populations as well as for testing high-risk groups.
Stem cell biology and tumorigenesis may be closely linked, and stem cells may have a role in the etiology of cancer (Al-Hajj et al, 2004; Al-Hajj et al, 2003; Lapidot et al, 1994; Pardal et al, 2003; Reya et al, 2001; WO 03/050502). Stem cells and tumor cells have many common features, including self-renewal, multi-drug resistance, telomerase expression and, in the instance of the prostate, androgen independence. It has been reported in WO 03/050502 that a small percentage of tumorigenic cells within an established solid tumor have the properties of stem cells. These solid tumor stem cells give rise both to more solid tumor stem cells and to the majority of cells in the tumor, cancer cells that have lost the capacity for extensive proliferation and the ability to give rise to new tumors. Thus, solid tumor cell heterogeneity reflects the presence of a variety of tumor cell types that arise from a solid tumor stem cell.
Prostatic stem cells do not require androgens for survival, as evidenced by completely normal prostatic regeneration after more than 30 cycles of androgen ablation and supplementation, which results in involution and normal regeneration of this gland (Isaacs, 1985). As prostatic carcinoma usually progresses to an androgen-independent tumor (which may reflect a stem cell-like phenotype), an understanding of prostate cell biology is important for devising preventative or therapeutic approaches to prostate cancer.
In addition to being a target of carcinogenesis, prostatic stem cells may also be a potential source of benign prostate hyperplasia, or BPH (De Marzo et al, 1998). The isolation of these cells would therefore be likely to increase our understanding not only of normal prostate physiology but also of two of the most common diseases afflicting men, namely prostatic carcinoma and BPH.
The murine prostate consists of a branched ductal network with each duct consisting of a proximal region (adjacent the urethra), an intermediate region, and a distal region. Actively proliferating cells (transit amplifying cells) are located in the distal region of the ducts (Cunha et al, 1987a). The present inventors have previously shown that the proximal region of mouse prostatic ducts is enriched in a subpopulation of epithelial cells that have a number of properties of stem cells: they are slow-cycling, possess high in vitro proliferative potential, and single cells are able to reconstitute complex, highly branched glandular structures in vitro that contain basal and luminal cells (Tsujimura et al, 2002). In addition, cell digests from the proximal region contain cells that form significantly more prostatic tissue in an in vivo transplantation model than cells isolated from other prostatic regions. Furthermore, cell digests obtained from this transplanted tissue are again able to give rise to prostatic tissue when re-inoculated into new animals, confirming the location of prostatic stem cells in the proximal region.
It would be useful to isolate a subpopulation of cells in the prostate that have the attributes of stem cells, particularly because a characteristic of stem cells is their ability to engraft and proliferate in their “niche” within their compartment.
As stem cells in other organs have been identified by their expression of specific antigens, it would be useful to determine whether these antigens could be used to identify the prostatic stem cell population in the proximal region of ducts.
Isolation of prostatic stem cells and the elucidation of their phenotype would make it possible to examine their biology and their regenerative capacity. In addition, the relationship between prostatic stem cells and two common diseases of the prostate, benign prostatic hyperplasia (BPH) and prostate carcinoma could be studied, as both diseases may arise from prostatic stem cells (De Marzo et al, 1999). It has recently been proposed that stem cells are the cells most likely to accumulate mutations that result in neoplasia, and that tumors may contain a stem cell reservoir that can self-renew indefinitely (Reya et al, 2001; Passegue et al, 2003).
The phenotype of acute myelogenous leukemia cells is similar to that of hematopoietic stem cells (Bonnet et al, 1997), and tumorigenic breast cancer cells also resemble normal early multipotent breast epithelial cells (Al-Hajj et al, 2003). Therefore, it would be useful to isolate prostatic stem cells in order to permit an understanding of prostatic epithelial biology, which is relevant as the evolution of androgen-independent prostate carcinoma may reflect a stem-like state of the tumor (Reya et al, 2001; Passegue et al, 2003). Moreover, stem cells and tumor cells have many common features, including infinite life span, androgen independence, multi-drug resistance, and telomerase expression. Therefore, isolation and characterization of special features of these stem calls may make it possible to design rational therapies to treat prostate carcinoma and BPH.